Sign up to receive free email alerts when patent applications with chosen keywords are publishedSIGN UP

Abstract:

The present invention is generally directed to novel functionalized
biomolecules and methods for generating such biomolecules. Biomolecules
may generally include nucleic acids, peptides, multicomponent molecular
complexes and/or any other molecular products that may be produced by
living organisms. The present invention is further directed to cells
and/or organisms manipulated to produce such functionalized biomolecules.
The cells contemplated by the present invention include both prokaryotic
as well as eukaryotic cells. The functionalized biomolecules are produced
via materials introduced into the cell using standard molecular biology
techniques or are incorporated within the genomic nucleic acid of a cell
by standard recombination techniques. Further contemplated is the use of
such cells for sequestration of target molecules within the cells.

Claims:

1. An expression vector comprising: a chimeric gene encoding a peptide,
operatively linked to a functional promoter, said expression vector when
inserted into a host transcribes said chimeric gene into a gene product
which is translated into said peptide which is capable of binding to or
altering at least one target molecule which induces stress on said host;
wherein said peptide comprises a non-naturally occurring, randomly
selected peptide which is not derived from a naturally occurring peptide
and is selected by sequential culturing of a host exposed to said target
molecule and identifying conferred resistance to said stress on said host
caused by said target molecule.

2. The expression vector of claim 1, wherein said vector further
comprises at least one of a selection marker or a marker for selective
induction.

3. The expression vector of claim 2, wherein said vector further
comprises at least one selection marker and wherein said selection marker
is an antibiotic resistance marker.

4. The expression vector of claim 1, wherein said promoter is a T7 RNA
polymerase or a ribosomal RNA promoter.

5. The expression vector of claim 1, wherein said biomolecule ligand
includes an amino acid sequence selected from the group of SEQ ID NO 1,
MSHAYFVCNRCDSSNHSAHE or SEQ ID NO 2, MSHATATPASRRRLPLRS.

6. The expression vector of claim 1, wherein said at least one target
molecule is a waste fluid contaminant.

7. The expression vector of claim 6, wherein said waste fluid contaminant
is at least one of an inorganic molecule, an organic molecule, a toxin, a
peptide, or a viral particle.

8. The expression vector of claim 1, wherein said at least one target
molecule is at least one of hormones, antibodies, proteins, enzymes,
pharmaceuticals or metals.

9. An isolated cell comprising said expression vector of claim 1.

10. The cell of claim 9, wherein said cell is a prokaryotic cell or a
eukaryotic cell.

11. An isolated cell comprising: at least one artificially inserted
nucleic acid sequence encoding mRNA, said mRNA encoding a peptide which
binds to or catalytically alters a target molecule which induces stress
on said cell; wherein said at least one nucleic acid sequence is selected
by sequential culturing of cells containing said sequence with exposure
to at least one target molecule and identifying conferred to resistance
to said stress caused by said target molecule.

12. The cell of claim 11, wherein said peptide includes an amino acid
sequence selected from the group of SEQ ID NO 1, MSHAYFVCNRCDSSNHSAHE or
SEQ ID NO 2, MSHATATPASRRRLPLRS.

13. The cell of claim 11, wherein said cell is a prokaryotic cell or a
eukaryotic cell.

14. The cell of claim 11, wherein said target molecule is a waste fluid
contaminant.

15. The cell of claim 14, wherein said waste fluid contaminant is an
inorganic molecule, an organic molecule, a toxin, a peptide, or a viral
particle.

16. The cell of claim 11, wherein said target molecule is a hormone, an
antibody, a protein, an enzyme, or a metal.

17-20. (canceled)

21. A set of isolated cells comprising: a plurality of cells, each of
said cells comprising at least one artificially inserted nucleic acid
sequence encoding mRNA; wherein each of said plurality of cells comprises
a different artificially inserted nucleic acid sequence encoding mRNA, at
least one of which encodes a non-naturally derived peptide which binds to
a desired target molecule and confers an identifiable resistance to
stress induced by said desired target molecule to one of said plurality
of cells.

23. The set of isolated cells of claim 21, wherein said non-naturally
derived peptide comprises a non-antibody peptide sequence.

24. The set of isolated cells of claim 21, wherein said resistance to
stress caused by said desired target molecule comprises survivability of
at least one of said plurality of cells when exposed to said desired
target molecule.

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. provisional patent
application Ser. No. 60/917,961, filed May 15, 2007, entitled
"STRESS-DRIVEN IN VIVO SELECTION OF RNAs WITH USEFUL PROPERTIES", the
entire contents of which are hereby incorporated by reference. This
application is a continuation-in-part of U.S. utility patent application
Ser. No. 12/044,737, filed Mar. 7, 2008, entitled "FUNCTIONAL NUCLEIC
ACIDS FOR BIOLOGICAL SEQUESTRATION", the entire contents of which are
hereby incorporated by reference.

SEQUENCE LISTING

[0002] The peptide sequences MSHAYFVCNRCDSSNHSAHE and MSHATATPASRRRLPLRS,
titled SEQ ID NO 1 and SEQ ID NO 2, respectively, are hereby incorporated
by reference to the ASCII text file entitled "PSEQ1P1018US01_ST25.txt",
created May 14, 2008, of 663 bytes in size.

BACKGROUND OF THE INVENTION

[0003] Directed evolution of natural microbial populations has been shown
to be an efficient approach to study molecular mechanisms of natural
selection, adaptation and speciation. The short generation time, large
population size, simple life cycle, and ease of maintenance and storage
make bacterial and viral systems exceedingly suitable for evolution
experiments. Nevertheless, such experiments typically require multiple
iterations of the mutation-selection cycle that implies (1)
diversification of parental genetic material by spontaneous or induced
mutagenesis, and (2) selective amplification of successful genotypes
through differential reproduction of the microorganisms under defined
environmental constraints. At the end of the procedure, the acquired
genetic changes can be examined and related to those phenotypic features,
which differentiate the evolved cell lineages from the ancestral strain.

[0004] A simple way to direct the evolution of a microbial population is
to make it propagate under an appropriately applied stress.
Stress-induced imbalances in cellular metabolism result in reduced
fitness of the wild type lineage. At the same time, some of the emerging
mutants may exhibit a substantial tolerance of the harmful factor. During
prolonged cultivation under stressful conditions, these resistant
phenotypes will gradually substitute the wild type. Accordingly, the
population will drift towards higher frequencies of the mutated genes
associated with the resistant clones. The original genotype will
eventually be replaced with a new one, which confers an improved fitness
on the microbes exposed to the hostile environment.

SUMMARY OF THE INVENTION

[0005] The present invention is generally directed to novel functionalized
biomolecules and methods for generating such biomolecules. Biomolecules
may generally include nucleic acids, peptides, multicomponent molecular
complexes and/or any other molecular products that may be produced by
living organisms. The present invention is further directed to cells
and/or organisms manipulated to produce such functionalized biomolecules.
The cells contemplated by the present invention include both prokaryotic
as well as eukaryotic cells. The functionalized biomolecules are produced
via materials introduced into the cell using standard molecular biology
techniques or are incorporated within the genomic nucleic acid of a cell
by standard recombination techniques. Further contemplated is the use of
such cells for sequestration of target molecules within the cells.

[0006] Provided herein are embodiments of an expression vector comprising
a chimeric gene encoding selective biomolecule ligands capable of binding
to or altering target molecules, operatively linked to a functional
promoter, where the vector when transfected in a host transcribes the
chimeric gene.

[0007] Also, disclosed are embodiments of an isolated cell comprising the
expression vector described supra.

[0008] Additionally, disclosed are embodiments of an isolated cell
comprising at least one nucleic acid sequence, incorporated into a
genomic nucleic acid, where the nucleic acid encodes a biomolecule that
binds to or catalytically alters a target molecule.

[0009] Also provided herein are methods for sequestering within a cell a
plurality of target molecules, present in a bulk volume comprising,
generating a library of nucleic acid sequences coding for biomolecules
capable of binding to said target molecules; incorporating the nucleic
acid sequences in at least one nucleic acid within a cell; culturing the
cell to achieve a cell population; contacting the cell population with
the bulk volume; and separating the cell population from the bulk volume.
Furthermore, provided are methods for bioremediation of contaminants
present in a bulk volume further comprising, generating a library of
biomolecule ligands capable of binding to and/or altering target
molecules.

[0010] Other objects, features, and advantages of the present invention
will be apparent to one of skill in the art from the following detailed
description and figures.

[0011] The present invention together with the above and other advantages
may best be understood from the following detailed description of the
embodiments of the invention illustrated in the drawings.

BRIEF DESCRIPTION OF THE FIGURES

[0012] FIG. 1 illustrates an example of an expression vector of the
present invention;

[0014] FIG. 2a shows a hypothetical model of a peptide of the present
invention.

DETAILED DESCRIPTION OF THE INVENTION

[0015] The detailed description set forth below is intended as a
description of the presently exemplified device provided in accordance
with aspects of the present invention and is not intended to represent
the only forms in which the present invention may be practiced or
utilized. It is to be understood, however, that the same or equivalent
functions and components may be accomplished by different embodiments
that are also intended to be encompassed within the spirit and scope of
the invention.

[0016] Unless defined otherwise, all technical and scientific terms used
herein have the same meaning as commonly understood to one of ordinary
skill in the art to which this invention belongs. Although any methods,
devices and materials similar or equivalent to those described herein can
be used in the practice or testing of the invention, the exemplified
methods, devices and materials are now described.

[0017] "Biomolecule" refers generally to molecules of biological origin,
such as, for example, nucleic acids, peptides, combinations and complexes
thereof, and/or other appropriate biologically generated molecules.
Biomolecule may also refer to the both an expression vector encoding a
functional product and the functional product itself.

[0018] An "aptamer" refers to a biomolecule that is capable of binding to
a particular molecule of interest with high affinity and specificity. The
binding of a ligand to an aptamer, which may be a nucleic acid such as
RNA or DNA, or a combination thereof, or a peptide sequence, may also
change the conformation of the aptamer. This type of interaction, with a
small molecule metabolite, for example, coupled with subsequent changes
in aptamer function where the aptamer is RNA, has been referred to as a
`riboswitch`. Aptamers may also comprise non-natural nucleotides,
nucleotide analogs, non-natural amino acids and/or amino acid analogs.
The method of selection may be by, but is not limited to, affinity
chromatography and the method of amplification by reverse transcription
(RT), polymerase chain reaction (PCR) and/or any other appropriate
amplification method.

[0019] Aptamers have specific binding regions which are capable of forming
complexes with an intended target molecule in an environment wherein
other substances in the same environment are not complexed to the
aptamer. The specificity of the binding is defined in terms of the
comparative dissociation constants (Kd) of the aptamer for its ligand as
compared to the dissociation constant of the aptamer for other materials
in the environment or unrelated molecules in general. Typically, the Kd
for the aptamer with respect to its ligand will be at least about 10-fold
less than the Kd for the aptamer with unrelated material or accompanying
material in the environment. Even more preferably, the Kd will be at
least about 50-fold less, more preferably at least about 100-fold less,
and most preferably at least about 200-fold less.

[0020] A nucleic acid aptamer will typically be between about 10 and about
300 nucleotides in length. More commonly, an aptamer will be between
about 30 and about 100 nucleotides in length. A peptide aptamer will
typically be between 10 and about 100 amino acid residues in length and
more typically between 10 and 30.

[0021] The terms "nucleic acid molecule" and "polynucleotide" refer to
deoxyribonucleotides or ribonucleotides and polymers thereof in either
single- or double-stranded form. Unless specifically limited, the term
encompasses nucleic acids containing known analogues of natural
nucleotides which have similar binding properties as the reference
nucleic acid and are metabolized in a manner similar to naturally
occurring nucleotides. Unless otherwise indicated, a particular nucleic
acid sequence also implicitly encompasses conservatively modified
variants thereof (e.g., degenerate codon substitutions) and complementary
sequences and as well as the sequence explicitly indicated. Specifically,
degenerate codon substitutions may be achieved by generating sequences in
which the third position of one or more selected (or all) codons is
substituted with mixed-base and/or deoxyinosine residues (Batzer et al.,
Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem.
260:2605-2608 (1985); Cassol et al. (1992); Rossolini et al., Mol. Cell.
Probes 8:91-98 (1994)). Also included are molecules having naturally
occurring phosphodiester linkages as well as those having non-naturally
occurring linkages, e.g., for stabilization purposes. The nucleic acid
may be in any physical form, e.g., linear, circular, or supercoiled. The
term nucleic acid is used interchangeably with oligonucleotide, gene,
cDNA, and mRNA encoded by a gene.

[0022] A riboswitch is typically considered a part of an mRNA molecule
that can directly bind a small target molecule, and whose binding of the
target affects the gene's activity [Tucker B J, Breaker R R (2005).
"Riboswitches as versatile gene control elements". Curr Opin Struct Biol
15 (3): 342-8]. Thus, an mRNA that contains a riboswitch is directly
involved in regulating its own activity, depending on the presence or
absence of its target molecule. By definition, then, a riboswitch has a
region of aptamer-like affinity for a separate molecule. Thus, in the
broader context of the instant invention, any aptamer included within a
non-coding nucleic acid could be used for sequestration of molecules from
bulk volumes. Downstream reporting of the event via "(ribo)switch"
activity may be especially advantageous. A similar concept is coined by
the phrase "aptazyme" in which an aptamer region is used as an allosteric
control element and coupled to a region of catalytic RNA (a "ribozyme" as
described below).

[0023] A ribozyme (from ribonucleic acid enzyme, also called RNA enzyme or
catalytic RNA) is a RNA molecule that catalyzes a chemical reaction. Many
natural ribozymes catalyze either the hydrolysis of one of their own
phosphodiester bonds, or the hydrolysis of bonds in other RNAs, but they
have also been found to catalyze the aminotransferase activity of the
ribosome. More recently it has been shown that catalytic RNAs can be
"evolved" by in vitro methods [1. Agresti J J, Kelly B T, Jaschke A,
Griffiths A D: Selection of ribozymes that catalyse multiple-turnover
Diels-Alder cycloadditions by using in vitro compartmentalization. Proc
Natl Acad Sci USA 2005, 102:16170-16175; 2. Sooter L J, Riedel T,
Davidson E A, Levy M, Cox J C, Ellington A D: Toward automated nucleic
acid enzyme selection. Biological Chemistry 2001, 382(9):1327-1334.].
Winkler et al. have shown [Winkler W C, Nahvi A, Roth A, Collins J A,
Breaker R R: Control of gene expression by a natural
metabolite-responsive ribozyme. Nature 2004, 428:281-286.] that, similar
to riboswitch activity discussed above, ribozymes and their reaction
products can regulate gene expression. In the context of the instant
invention, it may be particularly advantageous to place a catalytic RNA
or ribozyme within a larger non-coding RNA such that the ribozyme is
present at many copies within the cell for the purposes of chemical
transformation of a molecule from a bulk volume. Furthermore, encoding
both aptamers and ribozymes in the same non-coding RNA may be
particularly advantageous.

[0024] The term "gene" is used broadly to refer to any segment of DNA
associated with a biological function. Thus, genes include coding
sequences and/or the regulatory sequences required for their expression.
Genes can also include nonexpressed DNA segments that, for example, form
recognition sequences for other proteins. Genes can be obtained from a
variety of sources, including cloning from a source of interest or
synthesizing from known or predicted sequence information, and may
include sequences designed to have desired parameters.

[0025] As used herein, the term "bases" refers to both the
deoxyribonucleic and ribonucleic acids. The following abbreviations are
used, "A" refers to adenine as well as to its deoxyribose derivative, "T"
refers to thymine "U" refers to uridine, "G" refers to guanine as well as
its deoxyribose derivative, "C" refers to cytosine as well as its
deoxyribose derivative. A person having ordinary skill in this art would
readily recognize that these bases may be modified or derivatized to
optimize the methods of the present invention.

[0026] As used herein, the term "amino acids" refers to the 20 naturally
occurring amino acids used in peptide synthesis and the residues thereof
when present in a peptide molecule. A person having ordinary skill in
this art would readily recognize that these amino acids may be modified,
derivatized and/or supplemented by artificial amino acids or analogs
thereof to optimize the methods of the present invention. Amino acids may
be abbreviated as follows: A is alanine, R is arginine, N is asparagine,
D is aspartic acid, C is cysteine, E is glutamic acid, Q is glutamine, G
is glycine, H is histidine, I is isoleucine, L is leucine, K is lysine, M
is methionine, F is phenylalanine, P is proline, S is Serine, T is
threonine, W is tryptophan, Y is tyrosine, and V is valine.

[0027] The present invention is generally directed to novel functionalized
biomolecules and methods for generating such biomolecules. Biomolecules
may generally include nucleic acids, peptides, multicomponent molecular
complexes and/or any other molecular products that may be produced by
living organisms. The present invention is further directed to cells
and/or organisms manipulated to produce such functionalized biomolecules.
The cells contemplated by the present invention include both prokaryotic
as well as eukaryotic cells. The functionalized biomolecules are produced
via materials introduced into the cell using standard molecular biology
techniques or are incorporated within the genomic nucleic acid of a cell
by standard recombination techniques. Further contemplated is the use of
such cells and biomolecules, such as for sequestration of target
molecules within the cells.

[0028] In one aspect, the present invention includes a method of
generating selective biomolecule ligands in vivo. In one embodiment, a
library of diverse nucleic acid sequences is utilized in vivo for the
selection of functionalized biomolecules. The library of nucleic acid
sequences may be contained in an expression vector or vectors, each
comprising a chimeric gene encoding selective biomolecule ligands capable
of binding to or altering target molecules, operatively linked to a
functional promoter, where the vector when transfected and/or otherwise
introduced into a host organism or cell transcribes the chimeric gene.
The chimeric gene may generally code for a nucleic acid sequence which
may be transcribed into RNA. The transcribed RNA may be the product
biomolecule ligand of the expression vector and/or it may be a functional
messenger RNA (mRNA) which may subsequently bind to a ribosome where it
may be translated into a peptide, which may be the product biomolecule
ligand. In general, the method may comprise the steps of: [0029] 1)
Building populations of a host cell or organism carrying a replicate of
the expression vector; [0030] 2) Culturing said host populations under
specific conditions which may include selective stress, such as, for
example, the presence of a target molecule; [0031] 3) Assessing the
clones which display increased resistance to said specific condition; The
method may further comprise determining the specific sequence of the
expression vector after culturing and assessment as well as determination
of the functional activity of the biomolecule product of the expression
vector.

[0032] The chimeric gene generally includes a randomized, partially
randomized and/or selected sequence which may be evaluated for functional
activity in a host organism or cell. The chimeric gene may also be
subject to modifications and/or mutations after the determination of the
original sequence. The expression vector also generally includes a
promoter having high transcriptional activity such that the chimeric gene
is expressed at a high level in the host. The promoter may also include
functional elements such as inducible activity. In some exemplary
embodiments, the promoter is a T7 RNA polymerase promoter or a ribosomal
RNA (rRNA) promoter.

[0033] In some embodiments, the expression vector includes a chimeric gene
that encodes for and, when introduced into a host organism or cell,
transcribes mRNA and/or mRNAs. The mRNA may typically contain at least
one open reading frame (ORF) which may translate a peptide sequence when
associated with a ribosome. The ORF may generally be free of
unintentional interrupting stop codons, however stop codons may be
utilized to selectively interrupt translation. At least a portion of the
ORF is a randomized, partially randomized and/or selected sequence and
may have no obvious bias for a specific function, or it may be selected
for a known function. The encoded peptide may generally be a peptide
aptamer which may be selected for specific functional activity against a
target molecule, such as, for example, binding to or modifying a target
molecule. Target molecules contemplated include, but are not limited to,
metal ions, organic molecules, viral particles, biological molecules,
such as antibodies, proteins, enzymes, pharmaceuticals and/or any other
substance to be removed and/or treated from a bulk volume. In particular,
wastes and contaminants are contemplated. Sequestration of target
molecules refers to binding to or altering the target molecules.

[0034] Bulk volumes can be treated with the genetically modified cells
containing functional aptamer. The aptamer may be a peptide aptamer or a
nucleic acid aptamer. Further embodiments and methods for nucleic acids
are disclosed in detail in U.S. utility patent application Ser. No.
12/044,737, filed Mar. 7, 2008, entitled "FUNCTIONAL NUCLEIC ACIDS FOR
BIOLOGICAL SEQUESTRATION", the entire contents of which are hereby
incorporated by reference. The genetically modified cells may treat,
remove and/or sequester target molecules in the bulk volume. The presence
of a high concentration of binding and/or catalytic biomolecules inside
the cell creates an equilibrium shift in the bulk volume whereby a given
substance is removed from the bulk volume and sequestered in the cell by
binding to and/or catalytic action by the biomolecules. The sequestration
and/or catalytic action generally constantly removes the targeted
molecule from the equilibrium, resulting in a constant influx of the
target molecule into the cell. The genetically modified cells, harboring
the sequestered target molecules, are then removed from the bulk volume.
Appropriate methods of removal of the genetically modified cells include,
but are not limited to, filtration, sedimentation, centrifugation
(accelerated sedimentation), flocculation, adsorption, membrane
filtration, biofilm formation, membrane bioreactor, and/or any other
physical configuration otherwise known in the art as a bioreactor, used
to separate the treated waste stream from the cells.

[0035] Such bioreactors also include in situ remediation techniques in
which the genetically modified cells are released into a controlled
volume of the environment. Sequestration and/or chemical transformation
of contaminants then occurs before the controlled volume passes into
another portion of the environment. This is particularly useful in
examples where the cells are introduced into waste water and/or other
waste streams which are in contact with the environment. The genetically
modified cells can be immobilized for contact with a bulk volume while
not being distributed into the volume. Immobilization techniques include
but are not limited to, microbial mats, mineral amendments, polymer gel
formulations, and/or any other appropriate immobilization technique or
combination may be utilized. Genetically-modified cells can be tagged for
identification such that they can be isolated from a particular
environment. Additionally, the cells can be genetically modified to
include features for their removal from an environment, such as, for
example, a susceptibility factor to a particular substance, an affinity
to a particular separation method, and/or any other appropriate removal
method.

[0036] Further, the cells may also include features for increasing the
sequestration rate of a substance in a bulk volume. For example, a
molecular channel and/or transporter may be utilized to enhance transport
of a substance across the cell membrane into the cell. Metal ion and/or
other small ion transport molecules are known and can be incorporated by
genetic modification of the cell. Additionally, the cells can be
engineered to export the biomolecules into bulk environment, for example,
by including nucleic acid sequences encoding viral packaging and/or
export signals. Reuptake of biomolecule bound to the target molecule can
be engineered for example, by binding to cell surface receptors and/or
any other appropriate method.

[0037] Biomolecules as discussed above can be utilized as affinity handles
for purification. For example, biomolecule handle may be attached to a
molecule of therapeutic or diagnostic value, such as a peptide aptamer
affinity handle coupled to an antibody. The desired high-value molecule
is readily purified by binding the aptamer portion. Aptamers to common
chromatographic matrices such as agarose, Sephadex, Sepharose, as well as
more specialized affinity resins with immobilized metals, antibodies,
proteins, peptides, and/or any other appropriate affinity material can be
utilized. Aptamers to such affinity ligands are developed by well
established in vitro methods or by in vivo methods similar to those
discussed above. Inserted aptamers fused to desired molecules can be used
for therapeutic and/or diagnostic functions, such as, for example,
antibodies, hormones, signaling proteins, enzymes and/or any other
appropriate molecule. Aptamers utilized as affinity handles for molecules
can be sequenced, probed by hybridization, and/or characterized by some
other analytical technique, such as, for example, sequencing or mass
spectrometry for organism identification. In some embodiments, the
aptamer itself may be a desired molecule which may be purified by
affinity to its target molecule.

[0038] Inserted functional biomolecules are also useful for highly
specific intracellular labeling and/or cellular signal tracking. For
example, an aptamer including a fluorescent- and/or radio-label can be
concatenated and/or fused to an aptamer targeting a particular cellular
component, such as an important protein, enzyme, organelle, and/or any
other appropriate component. This aptamer fusion can be expressed at high
levels in a cell. Cells expressing such aptamers may thus have a built-in
ability to monitor specific cellular processes.

[0039] In yet another embodiment, there is provided a method for
sequestering within a cell a plurality of target molecules, present in a
bulk volume comprising, generating a library of nucleic acid sequences
coding for functional biomolecules binding to the target molecules;
incorporating the nucleic acid sequences into a cell; culturing the cell
to achieve a cell population; Contacting the cell population with the
bulk volume; and separating the cell population from the bulk volume. The
method further comprises recovering the target molecule from the cell
population. In general, the target molecules are inorganic molecules,
organic molecules, toxins, proteins, peptides, and viral particles. In a
related embodiment, the target molecules are hormones, antibodies,
proteins, enzymes, pharmaceuticals or valuable metals. In general, the
separation is accomplished by a method selected from the group consisting
of filtration, sedimentation, flocculation, adsorption, membrane
filtration, biofilm formation and membrane bioreactor interaction.

[0040] The following examples are given for the purpose of illustrating
various embodiments of the invention and are not meant to limit the
present invention in any fashion.

Example of Nucleic Acid Library Construction

[0041] A mini-gene library, as shown in FIG. 1, was designed to express a
20-mer peptide with 12 randomized positions in its central part and an
invariant four amino acid-long N- and C-terminus. The core element of the
library was a 100-bp dsDNA cassette, which was developed as a standard
module for composite genetic constructs with randomized segments. The
cassette contained a 60-bp ORF with a Shine-Dalgarno sequence located 8
by upstream of the translation start codon, XbaI and BamHI restriction
sites at the ends, and several auxiliary restriction sites (BsmAI, FokI,
and BspHI) that were intended for cassette modification and not used in
this study. Randomized segment of the ORF was presented as eleven
contiguous NNC triplets followed by one SRC triplet on the non-coding DNA
strand, where N corresponds to A, T, G, or C, S is G or C, and R is A or
G. The SRC triplet together with downstream invariant triplets encoded a
five amino acid-long C-terminal tag (H/R/D/G)SAHE, which was expected to
increase peptide stability in the E. coli cytoplasm. The total sequence
space of the library theoretically included 7.04×1013 DNA
variants, or 3.45×1013 peptide variants. The use of randomized
triplets with a fixed C in the third position inhibits the emergence of
internal stop-codons, equalizes distribution of different amino acids in
the peptide sequence, and significantly decreases the coding redundancy
of the library. It also diminishes the risk of a bias caused by an
occasional digestion of the randomized segment by restriction enzymes in
the course of library construction. On the other hand, this design
excludes methionine, tryptophan, lysine, glutamine and glutamic acid from
the variable part of the peptide. However, since none of the
above-mentioned amino acids possesses a unique functionality that cannot
be provided by other amino acids, they were considered to be dispensable
in this example. The cassette was inserted unidirectionally into
pCR21--T7pt vector between a T7 promoter and transcription terminator.
This resulted in a plasmid library designated as pCRRL1, which carries
ampicillin and kanamycin resistance determinants and requires
host-provided T7 RNA polymerase to express the randomized ORF. Placing
the randomized ORF under strong promoter on high copy number plasmid
ensures a very intense intracellular synthesis of the 20-mer peptide and
therefore was expected to contribute significantly to the modified E.
coli phenotype. It is noted that strong expression of the mini-gene might
be a source of metabolic stress, which would combine with the imposed
environmental one. However, when concentration of a bacteriotoxic
substance in the medium is close to lethal, the environmental stress is
likely to prevail and thus to direct the competition between clonal
lineages associated with different mini-gene variants.

[0042] The pCR21--T7pt plasmid was derived from cloning vector pCR2.1
(Invitrogen) by rearrangement of its MCS sequence and introduction of the
T7 late terminator, TΦ, downstream of the T7 RNA polymerase promoter
and MCS. The plasmid was cleaved at XbaI and BamHI sites, treated with
calf alkaline phosphatase, and gel purified. A 100 bp-long DNA cassette
harbouring an ORF with randomized sequence was synthesized in a single
reaction by overlap extension PCR from three deoxyoligonucleotides,
32-mer d(GCTCTAGAAGGAGATATACATATGTCTCACGC), 32-mer
d(CGGGATCCTAGGGATGTTATTCATGAGCGGAG), and 61-mer
d(CATATGTCTCACGCT(NNC)11SRCTCCGCTCATG), where N is an equimolar mixture
of A, T, G, or C, S is an equimolar mixture of G or C, and R is an
equimolar mixture of A or G. The PCR product was treated with XbaI and
BamHI restriction enzymes, gel purified, and ligated into linearised
dephosphorylated pCR21--T7pt plasmid (40 Units/μl of T4 DNA ligase,
0.04 Unit/μl of yeast inorganic pyrophosphatase in 1×NEB T4 DNA
ligase buffer, 100 hrs at 8-12° C.). The resulting plasmid
library, pCRRL1 (FIG. 1), was further purified by phenol-chloroform
extraction and ethanol precipitation. The size of the inserted DNA
fragment was verified by PCR with a pair of flanking primers, SP1 and SP2
(see above). No cassette oligomerization or unproductive re-circulization
of pCR21--T7pt was observed.

Example of Selection and Primary Characterization

[0043] The pCRRL1 library was transformed into E. coli BLR(DE3) cells,
which carry a T7 RNA polymerase gene under control of IPTG-regulated
lacUV5 promoter. The number of individual clones was estimated to be
˜105. The clone library was allowed to grow for 4 hrs in order
to obtain several replicates of it, and then split into parts intended
for parallel evolution experiments using different toxic agents.
Expression of the randomized ORF was induced by IPTG, and the multi-step
selection experiment was started by adding appropriate amounts of
NiCl2, AgNO3, or K2TeO3 to the cell cultures, as
shown in FIG. 2 with the selection of the E. coli clones resistant to
NiCl2 (a), AgNO3 (b), and K2TeO3 (c). Large filled
circles represent bacterial cultures successfully growing at indicated
concentrations of each toxic agent. Small open circles mark the
conditions under which the bacterial cultures did not exhibit noticeable
growth during a 24 hours selection cycle. Since no bacterial growth was
observed at AgNO3 concentrations above 0.9 mM and K2TeO3
concentrations above 18 mM, the corresponding data points are not shown
on plates (b) and (c), respectively. Black arrows point to the most
resistant bacterial cultures, from which individual clones have been
isolated for further studies of their toxicity tolerance. Grey arrow
marks the culture, which was used as a non-adapted control in tests of
the bacterial resistance towards nickel, silver, and tellurite. After
22-24 hrs of growth under stress, bacterial cultures exhibiting the
highest toxicity tolerance in each set were used as a seed for the next
cycle. A concentration gradient of the toxic agents was chosen to cover a
range within which an MIC was expected to be found. Thus, at every step
the selective pressure was adjusted depending on the achieved level of
the toxicity tolerance to keep the evolving bacterial populations under
subinhibitory conditions. Substantial increase in bacterial resistance to
Ni2+, Ag+, and TeO32- was observed during four, five,
and seven consecutive selection cycles, respectively. After that, no
further change in toxicity tolerance was detected. During the selection
procedure, the apparent MIC values increased from 4 to 10 mM NiCl2,
from 0.03 to 0.90 mM AgNO3, and from 0.7 to 20 μM
K2TeO3. Eight clones were isolated from each of the three
evolved resistant populations by plating. These were tested for the
dependence of growth on mini-gene expression by measuring OD600 after 24
hrs of cultivation in the presence of corresponding toxic agent, with and
without IPTG. Significant IPTG-induced growth improvement was observed
for several Ni2+-resistant clones while growth of Ag+- and
TeO32--resistant selectants was not affected by IPTG. Plasmid
sequencing revealed absolute sequence homogeneity within each group of
the studied clones. All eight Ni2+-resistant clones harbored the
same plasmid (designated as pCRRL1-N94-01) with a mini-gene variant
coding for the peptide MSHAYFVCNRCDSSNHSAHE, a structural model of which
is shown in FIG. 2a. In addition, a 9-bp deletion was detected in the
spacer region between the ORF and the transcription terminator. However,
since no important element of the mini-gene operon was located there, the
deletion was unlikely to affect the peptide expression.
Ag+-resistant clones were found to bear pCRRL1-A226-01 plasmid with
mini-gene variant coding for an 18-mer with the sequence
MSHATATPASRRRLPLRS. The shortening of the peptide was due to two
non-contiguous single-base deletions inside the ORF, which resulted in
frameshift and premature translation stop. Interestingly, the first
nucleotide in the transcribed part of the mini-gene operon was found to
be T instead of the original G. This can decrease the transcription
efficiency and disrupt a stability tag at the 5'-terminus of the
transcript, thus making it vulnerable to ribonucleolytic degradation. All
TeO32--resistant clones carried a version of plasmid
pCRRL1-T507-01 that has a 225-bp deletion of the whole mini-gene
transcriptional unit together with adjacent downstream sequence. This
deletion obviously abolishes T7 RNA polymerase activity on the plasmid,
making it irresponsive to IPTG induction.

Example of Assessment of Mini-Gene Dependent Toxicity Tolerance

[0044] The existence of a positive correlation between the level of
mini-gene expression and the extent of toxicity tolerance would strongly
argue in favor of a role for the mini-gene in the mechanism of stress
resistance of the evolved clones. Therefore, IPTG dependence of the
toxicity tolerance was explored to establish the importance of the
selected mini-gene variants for the improved bacterial performance under
stress. Comparison of the bacterial growth parameters measured in the
presence and absence of IPTG made it possible to distinguish the specific
effect of mini-gene expression on culture survival and propagation from
the basal resistance to the hostile environment acquired through adaptive
genomic mutations. In the case of TeO32--resistant clones, the
observed lack of IPTG influence on their growth coincides with the
deletion of the mini-gene operon from the plasmid. Thus, it is evident
that the evolved TeO32- tolerance must be wholly attributed to
mutations in the bacterial chromosome. The situation with the
Ag+-resistant selectants is less certain. The presence of a
seemingly functional mini-gene operon in the plasmid was nevertheless not
accompanied by an IPTG-induced stimulation of bacterial growth in
Ag+-containing medium. For three of the selected clones, the effect
of IPTG on the growth curves was studied more thoroughly at 0.02-0.8 mM
AgNO3 to confirm the results of the initial tests. Within the
accuracy of measurement no difference was found between cultures grown in
the presence and absence of IPTG. Transformation of the original E. coli
BLR(DE3) strain with plasmids isolated from the resistant clones did not
result in a phenotype with increased silver tolerance. Therefore, it was
concluded that either the peptide expressed from the mini-gene does not
contribute by itself to the evolved silver tolerance, or the expression
proceeds with very low efficiency. Among eight tested Ni2+-resistant
selectants, four exhibited two- to fourfold IPTG-induced increase in
OD600 measured after 24 hrs of cultivation in 9 mM
NiCl2-containing medium. For the rest of the clones, only marginal
growth stimulation by IPTG was observed under the same conditions. To
elucidate such a diverse behavior, two clones representing the
extremities of bacterial response to IPTG, N9405 and N9408, were taken
for a detailed study of IPTG influence on their growth under nickel
stress. It was found that IPTG significantly stimulates the growth of
both clones when the concentration of NiCl2 in the medium was 4 mM
and above. At lower nickel concentrations, IPTG provided little
stimulation, or even inhibited bacterial growth. Despite generally
similar reaction to the IPTG induction, the clones apparently differed in
their growth parameters, especially in the duration of the lag phase. It
was concluded that the mini-gene is active in both selectants but its
actual contribution to the evolved nickel tolerance is likely to be
modulated by differences in genetic background and may not be obvious
under some experimental conditions. In contrast to the resistant clones,
the non-adapted control culture was able to grow only when NiCl2
concentration in the medium was below 4 mM, and was always insensitive to
IPTG induction. Since the basal nickel tolerance of N9405 and N9408
clones was significantly higher than that of the control culture, it is
clear that they acquired adaptive genomic mutations during the selection
procedure.

[0045] The invention may be embodied in other specific forms without
departing from its spirit or essential characteristics. The described
embodiments are to be considered in all respects only as illustrative and
not restrictive. The scope of the invention is, therefore, indicated by
the appended claims rather than by the foregoing description. All changes
to the claims that come within the meaning and range of equivalency of
the claims are to be embraced within their scope. Further, all published
documents, patents, and applications mentioned herein are hereby
incorporated by reference, as if presented in their entirety.

Patent applications in class Library contained in or displayed by a micro-organism (e.g., bacteria, animal cell, etc.) or library contained in or displayed by a vector (e.g., plasmid, etc.) or library containing only micro-organisms or vectors

Patent applications in all subclasses Library contained in or displayed by a micro-organism (e.g., bacteria, animal cell, etc.) or library contained in or displayed by a vector (e.g., plasmid, etc.) or library containing only micro-organisms or vectors